Methods and compositions to modify the immunogenicity of a vascularized organ or tissue

ABSTRACT

Disclosed herein is an RGD-enriched solubilized extracellular matrix composition derived from endothelial cell culture that can be used to modify the immunogenicity or thrombogenicity of an organ intended for transplant. The RGD-enriched solubilized extracellular matrix composition is applied to the lumen of the vasculature of the organ, thereby placing a barrier between the antigens on the lumenal surfaces of the transplanted organ and the blood of the recipient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional applicationSer. No. 61/722,507 filed Nov. 5, 2012, the contents of which are hereinincorporated by reference into the present application.

FIELD OF THE INVENTION

The invention relates generally to transplantation of organs and inparticular to a method for reducing the immunogenicity of a graftintended for transplant.

BACKGROUND

Organ transplantation is the therapy of choice for endstage organfailure. In the case of kidney failure, for example, transplantationprovides for increased life expectancy, enhanced quality of life, and ismore cost-effective than maintaining patients on hemodialysis. In thecase of extrarenal organs, transplantation is life-saving since noequivalent to hemodialysis exists for these organs.

Rejection of the transplanted graft is an immunological response inwhich the recipient's immune system recognizes the graft as “foreign”and attempts to eliminate the transplanted graft. Part of the responseinvolves binding of the recipient's antibodies to the donor's vascularendothelial cells lining the blood vessels within the transplantedgraft. Antibody deposition leads to the activation of the complementcascade which mediates a cytotoxic phenomenon which can directly damageor kill the endothelial cells.

In addition the complement cascade leads to the activation of theendothelial cells which causes subsequent change in the anticoagulantenvironment. More specifically, the vascular endothelium normallyprovides a nonthrombogenic surface; however, when activated by theimmune system during the rejection process, the endothelial liningtransforms into a procoagulant environment. The resultant prothrombotic(thrombogenic) surfaces then attract polymorphonuclear cells andplatelets, resulting in the endothelium being damaged and causingseparation from the underlying substratum, and ultimately, severethrombosis of the graft.

The standard approach to mitigating the rejection process is to treatthe transplant recipient daily with an immunosuppressive regimen.However, currently immunosuppressive regimens are systemic; i.e., inaddition to suppressing immune function against the transplanted graft,immune function which protects the recipient from other processes (suchas infections) is suppressed. Further, the currently availableimmunosuppressants may cause substantial non-specific, toxic effects oncell types other than cells of the immune system.

U.S. Pat. No. 5,643,712 describes a method for treating and renderinggrafts nonthrombogenic and substantially nonimmunogenic using anextracellular matrix coating. Disclosed is a material that can beapplied to the luminal surface of the blood vessels of a vascularizedtissue or organ.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a composition and methodfor treating luminal surfaces of the vasculature of a tissue or organfor the purpose of rendering the tissue or organ substantiallynon-immunogenic and non-thrombogenic, the method comprising (a)establishing the organ or tissue in a warm perfusion system capable ofsupporting the near normal oxidative metabolism of the organ or tissue;(b) perfusing the organ or tissue with a non-blood perfusion solution,containing a component of the citric acid cycle selected from coenzymeA, FAD, DPN, cocarboxylase and TPN, for a time sufficient forvasodilation to occur; and (c) introducing into the organ or tissue asolubilized sonicated extracellular matrix preparation in an amountsufficient to substantially coat the luminal surfaces of the vasculatureof the organ or tissue, wherein said solubilized sonicated extracellularmatrix preparation renders the organ or tissue nonthrombogenic, andsubstantially nonimmunogenic.

In one aspect the invention relates to an Arg-Gly-Asp (RGD)-enrichedsolubilized extracellular matrix comprising extracellular matrix proteinfrom cultured endothelial cells, wherein said protein in said matrix isin the amount of 1-5 mg/ml and has an RGD absorbance at 450 nm of 0.3 to0.7, and wherein said extracellular matrix has a fragment size of lessthan 2 microns.

In a related aspect, the invention relates to a method for making anRGD-enriched solubilized extracelluar matrix composition, the methodcomprising culturing endothelial cells to confluence in an endothelialcell culture medium comprising ascorbic acid, retinoic acid and aglucose concentration in the range of about 1 to about 10 mg/ml; (b)decellularizing the culture to remove endothelial cells but leave theintact extracellular matrix; (c) solubilizing the extracellular matrixby acidification in the cold; (d) disrupting the solubilizedextracellular matrix to ensure fragment size of <2 microns; and (e)recovering the RGD-enriched solubilized extracellular matrix, whereinsaid solubilized extracellular matrix comprises extracellular proteinfrom endothelial cells in the amount of 1-5 mg/ml, an RGD absorbance at450 nm of 0.3 to 0.7 and a fragment size of less than 2 microns. In oneembodiment, the concentration of ascorbic acid in the culture medium isabout 20 μg/ml, the concentration of retinoic acid is about 152 μg/mland the glucose concentration about 5 mg/ml.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a and b are electron micrographs of vascular endothelial cells(VECs) lining the luminal surface of the renal arcuate artery before (a)and after (b) administration of the composition of the invention.

FIG. 2 shows the results of experiments to evaluate the effect of ECM onantigen presentation and T-cell activation.

FIG. 3 shows binding of an anti-β1 integrin antibody to a monolayer ofvascular endothelial cells as an indication of the effectiveness of theECM preparation of the invention compared to prior preparations.

DETAILED DESCRIPTION OF THE INVENTION

All patents, published patent applications, and non-patent referencescited herein, including U.S. Pat. Nos. 6,642,045, 6,582,953 and6,375,613 are hereby incorporated by reference in their entirety intothe subject application.

In the description that follows, certain conventions will be followed asregards the usage of terminology.

The term “organ, tissue or section of anatomy” refers to an excisedviable and whole section of the body to be maintained as such in the EMSof this invention, and refers to an intact organ including, but notlimited to, a kidney, heart, liver, lung, small bowel, pancreas, brain,eye, skin, limb or anatomic quadrant. The term “organ product” refers toany substance generated as the result of the secretory function of anorgan, frequently a fluid, for example, bile from liver, urine fromkidneys, but also includes mechanical functions such as kidneyfiltration or heart pumping.

The terms “perfusion solution” and “perfusate” are used interchangablyand refer to a non-blood buffered physiologic solution that providesmeans for reestablishing cellular integrity and function in organs whichmay have experienced ischemic damage prior to or during isolation andfurther, enables an organ or tissue to be maintained at a near normalrate of metabolism.

The term “non-blood” is intended to exclude perfusates comprisingsubstantially whole blood or its individual components. The perfusionsolution of the present invention may, however, contain a minimal amountof whole blood or a blood component, for example, red blood cells, serumor plasma.

The terms “near normal rate of metabolism” and “near normal metabolicrate” are defined as about 70-100% of the normal rate of metabolism fora particular organ as determined by measuring and evaluating whetherfunctional characteristics of an organ, such as those described in U.S.Pat. No. 5,699,793, are within the range associated with normal functionfor that particular organ. Examples of functional characteristicsinclude, but are not limited to, electrical activity in a heart asmeasured by electrocardiogram; physical and chemical parameters of organproduct, for example, oxygen consumption and glucose utilization whichcan be ascertained from perfusate concentrations; pancreatic enzymes;heart enzymes; creatinine clearance and filtration functions, andspecific gravity of urine and so on.

The present invention provides an optimized material and method formodifying the immunogenicity and thrombogenicity of tissues and organs.The immunocloaking treatment protects transplanted tissues and organsfrom allorecognition that normally occurs when circulation of therecipients' blood through the implanted tissue or organ is resumed.Recipient immune cells in circulating blood recognize the transplantedtissue as foreign. The interface of the subsequent immune-mediatedrejection is the vascular endothelium, i.e. the point where therecipient's immune cells meet donor tissue. The vascular endothelium isthe target of immune rejection whether the response is immediate,accelerated, acute or even chronic. The ability to adequately modify theimmunogenicity of the vasculature within an allograft prevents theimmune rejection cascade.

Immunocloaking Material

U.S. Pat. No. 5,643,712 describes an extracellular matrix materialproduced by culturing corneal endothelial cells in flasks. When thecorneal endothelial cells reached a confluent state, that is, when theentire surface of the culture vessel was covered with cells with tightcell junctions, the flasks were decellularized non-enzymatically toremove the corneal endothelial cells while leaving the extracellularmatrix intact. The extracellular matrix was then solubilized byacidification in the cold. When acidified solubilized extracellularmatrix was neutralized at normothermic temperature, the solubilizedextracellular matrix components re-polarized to form a reconstitutedextracellular matrix. It was proposed that the neutralized extracellularmatrix components could be applied to the luminal surfaces of thevasculature during near-normothermic perfusion to result in a modifiedluminal vascular surface that was nonimmunogenic and nonthrombogenic.

Disclosed herein is an improved immunocloaking material. Theextracellular matrix preparation produced by the cultivation of cornealendothelial cells was optimized by culturing endothelial cells in atissue culture medium supplemented with a combination of ascorbic andretinoic acids and an increased carbohydrate concentration. Theresulting extracellular matrix that is produced using the enhancedmedium contains a higher concentration of proteins and an increasedconcentration of Arg-Gly-Asp (RGD) sequences in the proteins. RGDfacilitates binding to the surface of the vascular endothelial cells viatheir respective adhesion molecules. The extracellular matrix producedby the present invention predominantly consists of a network ofmolecules with self-assembly consisting of a laminin template for thescaffolding, glycoproteins, proteoglycans, carbohydrate moieties,vimentin, fibronectin, elastin and a network of collagen fibrils. Theability of the extracellular matrix to effectively bind to the luminalsurfaces of the vascular endothelial cells lining the vasculature isdependent upon interactions that are affected by the number andintensity of the adhesions between the vascular endothelial cell surfaceintegrins and specific peptides in the polymerized extracellular matrix.A number of surface integrins facilitate the vascular endothelial cellbinding to the extracellular matrix of the present invention. However,the β1 integrin sub-family represents a major class of integrins thatinteract with a number of the individual components of the extracellularmatrix of the present invention, including laminins and type IVcollagen. These vascular endothelial cell integrins provide for theanchorage of the extracellular matrix components that facilitates there-assembly of the solubilized and neutralized material that providesthe immunocloaking membrane. (132,133) In vitro studies using ECMprevented cell penetration into the engineered tissues while alsoproviding a compatible environment for confluent cell populations.

Cultivation of Corneal Endothelial Cells (CEC)

In one embodiment, the immunocloaking material of the invention isderived from corneal endothelial cells (CEO), which were obtained asfollows. Corneas were dissected from the intact orbit maintainingsterility. The luminal surface was treated with a digestive enzyme andincubated at 37° C. for 20 minutes. The digestate was collected, washedand resuspended in tissue culture media. The isolated cornealendothelial cells were then placed in tissue culture flasks and fedevery three days until confluent. Control cultures were maintained inMedium 199 supplemented with 10% serum and Fibroblast GrowthFactor-basic (FGF) (5 ng/ml). Experimental cultures were cultured in thesame medium as control cultures, but supplemented with ascorbic acid (20ug/ml), retinoic acid (152 μg/ml), glucose (5 mg/ml), VascularEndothelial Growth Factor (VEGF)(5 ng/ml), Epithelial Growth Factor(EGF)(5 ng/ml) and Insulin-like Growth Factor (0.5 ng/ml) (referred tohereinafter as the AA/RA-supplemented media). As shown in Table 1,culture of CECs in the AA/RA-supplemented media resulted insignificantly shorter times to confluence and a shortened populationdoubling time.

TABLE 1 Corneal Endothelial Cell (CEC) Growth Curves Primary CECcultures Control Media Present Invention Time to Confluence 7 days(+/−2) 2 days (+/−0.5)

Without wishing to be bound by theory, it is thought that retinoic acidfunctions by accelerating the induction of corneal endothelial celldifferentiation and by contributing to increased extracellular matrixsecretion. This increased secretion of extracellular matrix is in partattributable to increased laminin, collagen type IV andglycosaminoglycans synthesis. The higher concentration of glucoseprovides a positive effect on the metabolic rate and the correspondingrate of synthesis. Ascorbic acid provides an additive effect on thesynthesis of the various components of the extracellular matrix by thecorneal endothelial cells.

While a range of glucose can be used, most commonly used tissue culturemedia contain a normal glycemic concentration of 5.5 mM (1 g/L).Ascorbic acid is not usually included in the formulations of commontissue culture media and retinoic acid is rarely included.

Secreted Extracellular Matrix

Extracellular matrix from the corneal endothelial cells grown in thecontrol media was acidified, incubated and scraped from the surface ofthe tissue culture vessel. Similarly, the extracellular matrix producedby the corneal endothelial cells grown to confluence in theAA/RA-supplemented media was also recovered by acidification. Whenharvested, the solubilized extracellular matrix is not uniform and maycontain large fragments that are not easily broken apart. In order toadminister the solubilized extracellular matrix in a way that does notobstruct or clog smaller blood vessels and to also result in a uniformlayering of the immunocloaking matrix membrane it is necessary tofurther fragment the solubilized material. The large fragments can befurther reduced in size by several methods. These methods includesonification, pepsin digestion and guanidine-HCl treatment. Sonificationentails fragmentation that results in smaller particles sizes rangingfrom 5-20 nanometers. Care is needed to limit the unfolding of theprotein and the formation of amyloid-like structures. Pepsin digestionof the large fragments into peptides is effective in the acidicenvironment of the soluble extracellular matrix mixture. Guanidine-HClfractionates the large protein fragments in the acidified solubilizedextracellular matrix and allows for the re-polymerization whenneutralized and at near-normothermic temperatures.

Characteristics of the Immunocloaking Extracellular Matrix

The extracellular matrix produced by the corneal endothelial cells grownin AA/RA-supplemented media contains a significantly higherconcentration of protein in comparison to the control preparationsproduced by the method of U.S. Pat. No. 5,643,712 as measured using astandard Lowry assay (Table 2). The extracellular matrix wasdecellularized, acidified and then treated to render the solubilizedmaterial into small fragments. The protein concentration was determinedin batch of extracellular matrix manufactured by the method and processof the present invention (n=10) and compared to the material produced bythe method of U.S. Pat. No. 5,643,712A.

Using an antibody to the peptide sequence Arg-Gly-Asp (RGD) in anindirect immunofluorescence assay, the extracellular matrix produced bythe method and process of the present invention results in animmunocloaking membrane that contains significantly increased RGD incomparison to control extracellular matrix produced by the method ofU.S. Pat. No. 5,643,712A (Table 2).

Confluent cultures of corneal endothelial cells were decellularized andthe exposed extracellular matrix was tested by incubating the exposedmembrane with an antibody to the RGD-containing peptides after firsttreating the decellularized surface with a blocking step consisting of5% BSA for one hour. The matrix membranes were then washed with PBS andthen a secondary fluorescein tagged antibody was applied and incubatedfor an additional one hour. Following incubation, the matrix membranewas again washed and the absorbance at 450 nm was determined. Theresults are listed in Table 2.

TABLE 2 Analysis of the immunocloaking extracellular matrix ControlExtracellular Matrix Present Invention Protein concentration 518(+/−172) 4,918 (+/−1,425) (ug/ml) RGD*  0.1 (+/−0.05)  0.5 (+/−0.03)*absorbance at 450 nm

Alternatively, an optimized immunocloaking membrane can be obtained byisolating the naturally occurring sub-endothelial cell extracellularmatrix from human blood vessels. In particular, endothelial cells fromhuman umbilical cord veins (HUVEC) and arteries can be the source of arich extracellular matrix that displays the same immunocloakingproperties as the optimized extracellular matrix produced by cornealendothelial cells in culture. The native extracellular matrix can beobtained by decellularization of the vascular endothelium that lines theluminal surface of blood vessels in vivo. The extracellular matrix canthen be harvested by acidifying the blood vessel lumen and solubilizingthe sub-endothelial matrix. Following the collection of the nativeextracellular matrix, the material can be neutralized, stored andre-polymerized.

Fragmentation of the Extracellular Matrix

The isolated extracellular matrix material, whether from the culture ofcorneal endothelial cells or native blood vessels must be processed toensure that the components are fragments small enough to be applied tocomplex vasculature without clogging the microvessel beds. To adequatelydisperse the solubilized extracellular matrix constituents, it isnecessary to obtain fragments of <2 microns. This can be accomplished bya number of dispersion methods, for example, sonication of theextracellular matrix. Alternatively, the solubilized extracellularmatrix can be fragmented within an acceptable range by enzyme digestionor treatment with chaotropic agents such as guanine hydrochloride thatinterferes with the three dimensional structure of the protein(s) tohelp break apart large fragments. Once the solubilized extracellularmatrix is adequately fragmented to ensure even distribution andapplication within small diameter blood vessels, the mixture can beneutralized to a physiologic pH. By keeping the neutralizedextracellular matrix at a temperature range of 4° C. to 8° C.,re-polymerization will not occur. Re-polymerization will only occur whenthe mixture reaches a near-normothermic temperature.

Application of the ECM to the Vasculature

In order to uniformly apply the immunocloaking extracellular matrix tothe luminal surfaces of the vasculature of a vascularized tissue ororgan, the organ must first be established in a warm perfusion systemusing a substantially non-blood perfusate that is able to restore andsupport oxidative metabolism of the organ. Establishing the tissue ororgan in a warm perfusion system involves isolating an organ, tissue orspecific area of anatomy from the rest of the physiologic system byremoving or interrupting the arterial source of blood feeding thedesired tissue(s). Likewise, the venous outflow from the organ orsection of anatomy is interrupted and the venous effluent is collected.If the tissue is completely excised from the body, then the enervationand lymphatics of the tissue(s) are also isolated. Next, the organ ortissue is flushed through the arterial system with the solution of thepresent invention at a temperature of about 25°-37° C. to remove bloodand blood products. The organ is then placed in an exsanguinousmetabolic support system and perfused with non-blood perfusion solution,for example as described in U.S. Pat. No. 6,642,045, while variousparameters of the perfusion are monitored by the system and regulated asnecessary to maintain adequate metabolism of the organ or tissue. Organfunction is also monitored, for example, by collecting an organ product,such as urine or bile, and evaluating whether physical and chemicalparameters of the organ product are within the range associated withnormal function for that particular organ (see for example, U.S. Pat.No. 6,375,613, the contents of which are incorporated by reference.)

For purposes of the present method, the organ is perfused at anear-normothermic temperature using a non-blood perfusion solution for atime sufficient to restore oxidative metabolism and obtain NO-mediatedvasodilation of the vasculature of the organ, particularly themicrovessel bed, in order to avoid clogging the small diameter bloodvessels with the extracellular matrix material once it is introducedinto the organ. Without a continuous flux of NO and the resultingdilation, the immunocloaking material will clog the small diameter bloodvessels. In contrast, with adequate support of NO-mediated vasodilation,the neutralized but still solubilized cold immunocloaking material canbe uniformly applied to result in a continuous masking of the vascularendothelium along the vasculature (FIG. 1).

Establishment of Adequate Vasodilation Mediated By a Flux of NO

Maintenance of the organ in a warm perfusion system using a non-bloodperfusion solution enables the monitoring of NO flux to determine whenvasodilation of the vasculature is adequate and administration of theimmunocloaking material is appropriate.

Adequate vasodilation is usually achieved after about 30-minutes fromthe time that perfusion is initiated and will be presumed, for example,when mean arterial pressures are in the range of about 32 mmHg to about47 mmHg. Similarly, adequate vasodiation is usually accompanied by meanvascular flow rates in the range of about 90 cc/min to about 180 cc/min.

Immunocloaking material is infused into the organ or tissue at a rate ofapproximately 100 μg per minute. At a perfusion temperature of about 25°C. to 37° C., polymerization of the immunocloaking material will resultin a uniform covering several nanometers in thickness withoutobstruction of the blood vessel lumen. This unobstructed distributionoccurs also within the microvessel bed. The histologic findings indicatethat at a dose range of 66 to 660 μg per gram of kidney, more than 90%of the blood vessels within a vascularized tissue or organ can besufficiently and uniformly immunocloaked.

In order to obtain adequate and firm binding of the re-polymerizedextracellular matrix to the surface of the vascular endothelium it isnecessary to facilitate efficient integrin binding of the matrix via theRGD sequences within the immunocloaking material to the vascularendothelial cell family of integrins. To achieve this firm binding anadequate magnitude of oxidative metabolism must be maintained in theperfusing tissue or organ. A sufficient level of oxidative metabolism isnecessary in order to facilitate the requisite translocation of thevascular endothelial cell integrins that are normally localized at theabluminal surfaces of the cells. The translocation occurs upon signaltransduction initiated by the layering of the laminin components of thesolubilized extracellular matrix preparation. With adequate oxidativemetabolism as measured by oxygen consumption across the tissue or organof >0.1 cc/min/g, the translocated integrins are expressed on theluminal surface of the vascular endothelium. This translocation of thesurface integrins, in particular but not exclusively the β1 family ofintegrins, results in the firm binding of the re-polymerizedextracellular matrix to the vascular luminal surfaces of the bloodvessels within the tissue or organ. This firm binding persists followingreimplantation and reperfusion. Following such reimplantation andreperfusion the re-polymerized extracellular matrix remains intact alongthe luminal surfaces within the vasculature for a minimum of 21 daysrendering the tissues and organs nonimmunogenic and nonthrombogenic.

Competitive Binding Assay

A competitive binding assay was used to evaluate the binding capacity ofeach of the control and improved preparations of extracellular matrixmaterial. Vascular endothelial cells were grown to confluence in tissueculture wells. The confluent monolayer was washed three times. Theimmunocloaking material prepared in accordance with the method of U.S.Pat. No. 5,643,712 was compared for binding efficiencies to the materialproduced by the method of the present invention. The testing wasperformed in triplicate with 100 μl of each preparation added to theconfluent vascular endothelial cells. The extracellular matrixpreparation were incubated for 30 minutes at 37° C. and then copiouslywashed to remove any unbound extracellular matrix material. The wellswere then treated with anti-β1 integrin IgG and incubated again at 37°C. for 30 minutes. Following incubation, the wells were again copiouslywashed and a fluorescein tagged anti-IgG was added and the wells wereagain incubated for 30 minutes at 37° C. The labeled wells were thenread using a PerkinElmer VICTOR2 multilabel counter. The wells treatedwith the extracellular matrix made using the process of the U.S. Pat.No. 5,643,712 patent contained significantly more fluorescence than theextracellular matrix produced by the method of the present invention.(Table 3)

Immunocloaking Mechanism of Protection

The extracellular matrix produced, processed and applied as describedabove results in protection from the recipient's immune response for aminimum of thirty days. The application of the immunocloakingextracellular matrix membrane prevents allorecognition by the recipientby preventing antigen presentation and T cell activation. To demonstratethe impact of extracellular matrix ability to immunocloak, humanresponding mononuclear cells (responders) were stimulated withautologous lymphocytes (negative controls) and untreated confluentmonolayers of donor umbilical vein vascular endothelial cells (positivecontrols). The test groups consisted of the same confluent donorvascular endothelial cells that were immunocloaked with theextracellular matrix membrane of the present invention.

Early T cell activation was measured using CD4+, CD69+ detection by flowcytometry following 24 hours (n=10). Measurements were also made for thecytokines—IL-2, IL-6 and MIG using the Luminex xMap platform. Inaddition, following stimulation, supernatants were collected from eachtriplicate well and tested for additional cytokine and chemokineresponses to evaluate antigen presentation. These cytokines andchemokines tested associated with antigen presentation included: IL-1β,TNF-α, MIP-1α and γ-IFN.

Immunocloaking of the vascular endothelial cells resulted instatistically significant inhibition of the cytokines and chemokines:IL-1β, IL-6, γ-IFN, IL-2, TNF-α, CD-69, MIG and MIP-1α (p<0.005) (FIG.2). The finding of the inhibition of the cytokines produced by antigenpresenting cells, MIP-1α, IL-1β, γ-IFN and TNF-α suggest thatimmunocloaking vascular endothelial cells with the extracellular matrixmembrane of the present invention prevents antigen presentation. SinceIL-1, γ-IFN and TNF-α are pro-inflammatory cytokines, the ability of theextracellular matrix barrier membrane to inhibit their release providesfurther evidence of the protective effect of immunocloaking. Similarly,the inhibition of markers of T cell activation, IL-6, IL-2, CD-69 andMIG suggest the blockade of T cell mediated responses when the vascularendothelial cells are immunocloaked with the membrane. It is reasonableto assume that the immunocloaking extracellular matrix membrane thatprevents primary antigen recognition would also prevent endothelial cellactivation. Preventing endothelial cell activation would likewiseprevent the externalization of the pre-formed Weibel-Palade bodies thatcontain P-selectin adhesion molecules. Without a pro-inflammatory signalthe multi-step leukocyte extravasation process would be adverted whilethe extracellular matrix membrane masks the allograft. The results ofthese immunologic screenings support the hypothesis that immunocloakingcan be successfully used in an organ-specific manner to prevent theallorecognition that normally occurs upon reperfusion.

The present invention provides an optimized material and treatment thatcan be applied to the luminal surfaces within tissues and organs thatrenders the treated tissues nonimmunogenic and nonthrombogenic. Thematerial and its application can be used treat an allograft, tissueengineered construct or a xenograft. Such an immunocloaking therapy canprovide a window of protection that prevents the normal immune responseto engraftment of a foreign tissue. This window of opportunity providesa period where tolerance induction protocols can be successfullyapplied.

Re-Application of the Extracellular Matrix Membrane

Following the period of immune protection where antigen presentation andT cell activation and proliferation is prevented, it is feasible tore-administer the extracellular matrix membrane therapy. As the barriermembrane provided by the bound re-polymerized extracellular matrixdegrades, components of the membrane are exposed within the luminalsurfaces. A purified laminin scaffolding can be re-applied viaintroduction into the intravascular space and will bind to the surfaceof the deteriorating immunocloaking material. This can be followed byapplication of the complete solubilized extracellular material. The newlaminin scaffolding provides a substrate onto the deteriorating boundextracellular matrix membrane. The solubilized extracellular matrix thatis maintained cold and neutralized can then be re-administeredintravenously. The solubilized extracellular matrix preferentially bindsto the new laminin scaffolding and results in a newly formed boundextracellular matrix membrane that provides further protection fromimmune rejection. The re-administration of the extracellular matriximmunocloaking membrane can be reapplied approximately every 21 days.This replacement therapy that is tissue- and organ-specific cansubstitute for the daily multi-drug immunosuppressive regimens that areneeded today.

I claim:
 1. An Arg-Gly-Asp (RGD)-enriched solubilized extracellularmatrix comprising extracellular matrix protein from cultured endothelialcells, wherein said protein in said matrix is in the amount of 1-5 mg/mland has an RGD absorbance at 450 nm of 0.3 to 0.7, and wherein saidextracellular matrix has a fragment size of less than 2 microns.
 2. Amethod of modifying the immunogenicity and/or thrombogenicity of theluminal surfaces of the vasculature of a tissue or organ, the methodcomprising: (a) establishing the organ or tissue to be modified in awarm perfusion system capable of supporting near normal oxidativemetabolism of the organ or tissue; (b) perfusing the organ or tissuewith a non-blood perfusion solution comprising a component of the citricacid cycle selected from the group consisting of coenzyme A, flavinadenine dinucleotide (FAD), β-nicotinamide adenine dinucleotide (NAD orDPN), β-nicotinamide adenine dinucleotide phosphate (NADP⁺ or TPN⁺), andcocarboxylase, for a time sufficient for vasodilation to occur; and (c)introducing into the organ or tissue an Arg-Gly-Asp (RGD)-enrichedsolubilized extracellular matrix preparation comprising extracellularprotein from endothelial cell cultures in the amount of 1-5 mg/ml and anRGD absorbance at 450 nm of 0.3 to 0.7 in an amount sufficient tosubstantially coat the luminal surfaces of the vasculature of the organor tissue, wherein said solubilized extracellular matrix preparationrenders the organ or tissue nonthrombogenic, and substantiallynonimmunogenic.
 3. The method of claim 2, wherein the amount sufficientto substantially coat the luminal surfaces of the vasculature of theorgan or tissue to be modified is in the range of about 66 to about 660μg/gram weight of organ or tissue.
 4. The method of claim 2, whereinsteps b) and c) are performed in a warm preservation system at atemperature in the range of about 22° C. to about 35° C.
 5. The methodof claim 2, wherein the extracellular matrix preparation is derived fromhuman corneal endothelial cells.
 6. A composition comprising thesolubilized extracelluar matrix of claim 1
 7. A method for making anRGD-enriched solubilized extracelluar matrix composition, the methodcomprising: (a) culturing endothelial cells to confluence in anendothelial cell culture medium comprising ascorbic acid, retinoic acidand a glucose concentration in the range of about 1 to about 10 mg/ml;(b) decellularizing the culture to remove endothelial cells but leavethe intact extracellular matrix; (c) solubilizing the extracellularmatrix by acidification in the cold; and (d) disrupting the solubilizedextracellular matrix to ensure fragment size of <2 microns; and (e)recovering the RGD-enriched solubilized extracellular matrix, whereinsaid solubilized extracellular matrix comprises extracellular proteinfrom endothelial cells in the amount of 1-5 mg/ml, an RGD absorbance at450 nm of 0.3 to 0.7 and a fragment size of less than 2 microns.
 8. Themethod of claim 7, wherein the glucose concentration is between about2.5 and about 7.5 mg/ml.
 9. The method of claim 7, wherein the glucoseconcentration is between about 4 mg/ml and about 6 mg/ml.
 10. The methodof claim 7, wherein the glucose concentration is about 5 mg/ml.
 11. Themethod of claim 7, wherein the ascorbic acid concentration is betweenabout 10 and 30 μg/ml.
 12. The method of claim 7, wherein the ascorbicacid concentration is about 20 μg/ml.
 13. The method of claim 7, whereinthe retinoic acid concentration is between about 100 to 200 μg/ml. 14.The method of claim 7, wherein the retinoic acid concentration is about152 μg/ml.
 15. The method of claim 7, wherein said cells are endothelialcells.
 16. The method of claim 7, wherein said cells are cornealendothelial cells.
 17. The method of claim 7, wherein said cells arehuman umbilical vein endothelial cells (HUVEC).
 18. An RGD-enrichedextracellular matrix material obtained by the method comprising: (a)culturing endothelial cells to confluence in an endothelial cell culturemedium comprising ascorbic acid, retinoic acid and a glucoseconcentration in the range of about 1 to about 10 mg/ml; (b)decellularizing the culture to remove endothelial cells but leave theintact extracellular matrix; (c) solubilizing the extracellular matrixby acidification in the cold; and (d) disrupting the solubilizedextracellular matrix to ensure fragment size of <2 microns.
 19. Themethod of claim 18, wherein said endothelial cells are selected fromcorneal endothelial cells (CECs) and human umbilical vein endothelialcells (HUVEC).